The hallmark contribution of genetic engineering is technology that, when properly applied, can be used to elucidate precisely and alter in a controlled way—with base by base resolution—the genotype and thereby the phenotypic characteristics of living organisms. Two long-recognized applications of genetic engineering technology that stem from this contribution and promise great humanitarian, scientific and commercial benefits are the creation of improved organisms and the creation of organisms that can be used to produce substances other than those for which they are naturally useful.
As to the first of these goals, genetic engineering has been applied during the past two decades in a wide range of efforts to create improved organisms for food production. To mention just a few, these efforts include endeavors: to produce improved strains and varieties of agricultural plants; to produce livestock that utilize feed more efficiently; to endow livestock with greater resistance to parasites and disease; to create livestock that provides more healthful food; to alter livestock so that it is less harmful to the environment; and to produce plants and animals that benefit the environment. Regarding the production of transgenic farm animals, for instance, see Ebert (1989), Gene transfer through embryo microinjection, pgs 233-250 in ANIMAL BIOTECHNOLOGY: COMPREHENSIVE BIOTECHNOLOGY, FIRST SUPPLEMENT, Eds. Babiuk et al., Pergamon Press and, for another review in this regard, also see Ebert and Schindler (1993), Transgenic Farm Animals: Progress Report, Theriogenology 39: 121-135.
Towards the second goal, genetic engineering has been applied to the production of a wide variety of substances in plants and animals, most often proteins of pharmaceutical interest. These efforts largely have been directed, in animals, to the production of xeno-substances in the milk of livestock animals—primarily swine, ovine, caprine and bovine animals. Regarding the production of pharmaceuticals in milk see, for instance, Ebert and DiTullio (1995), The production of human pharmaceuticals in milk of transgenic animals, pgs 36-41 in THE NATURAL ENVIRONMENT: Interdisciplinary Views: Proceedings. Among efforts to produce substances in this way have been those aimed at the production of hormones, antibodies, enzymes, and factors involved in or related to hemostasis, to name just a few. These endeavors have met with varying degrees of success, as discussed below. Generally, complex polypeptides that undergo extensive post-translational modification have been produced successfully only in animals, and for the most part attempts to produce commercially valuable amounts of these products has been restricted to mammary gland expression and to isolation of proteins from milk.
Although none of these efforts have been entirely successful, several efforts to produce proteins transgenicly in milk have met with some success, and a number of proteins of pharmaceutical interest have been transgenicly expressed in mammary gland cells, and isolated from milk in a biologically active form. For an early review in this regard, see in pertinent part, for instance, Ebert and Schindler (1993), Transgenic Farm Animals: Progress Report, Theriogenology 39: 121-135. For instance, tPA has been produced in goat milk (see Ebert et al. (1991), Transgenic production of a variant of human tissue-type plasminogen activator in goat milk I: Generation of transgenic goats and analyses of expression, Bio/Technology 9: 835-838). And high levels of active human alpha-1-anti-trypsin have been produced in sheep milk (see Wright et al. (1991), High level expression of active human alpha-1-anti-trypsin in the milk of transgenic sheep, Bio/Technology 9: 830-834). Nevertheless, success has not been general and transgenic mammary gland-specific expression has not, as yet, been used to produce a pharmaceutical protein that has entered the marketplace.
Part of the reason for the overall difficulty in bringing these transgenic products to market may lie with disadvantages of present methods for transgenic production in milk. First, efficiency of transgenic production in mammary glands and milk is significantly reduced by gender specificity. Although males can be induced to lactate, they cannot be made to produce milk in quantities useful for commercial production of transgenic substances. See, for instance, Ebert et al. (1994), Induction of human tissue plasminogen activators in the mammary gland of transgenic goats, Bio/Technology 12: 699-702, and also Ebert (1988), A Moloney MLV somatotropin fusion gene produces biologically active somatotropin in a transgenic pig, Mol. Endoc. 2: 277-283. Practically all of the milk for commercial production of transgenic products using mammary gland-specific expression therefore must come from the females in a transgenic herd. Since, the quantities of milk useful to produce transgenic substances commercially therefore can be obtained only from the female “half” of a herd, milk based transgenic production methods are approximately 50% less efficient in utilizing a herd population than otherwise similarly efficient technology that is gender-neutral. Other aspects of milk-based transgenic production methods may make up for this relative disadvantage over other technologies; but, gender neutrality is nonetheless desirable, and would be an advantage even to milk based production.
Second, although lactation can be induced in immature animals of either sex, and milk thereby can be obtained in quantities sufficient to assess transgenic expression, there is nonetheless a delay from the time transgenic expression is proved in an immature animal to the time the animal can produce milk in quantities useful for commercial production of transgenic substances. In some species, moreover, the volume of milk produced simply is insufficient to support commercial production of proteins, at levels of expression and secretion that can be achieved in mammary gland cells. Even in mammals that produce the necessary volumes of milk, lactation often must be induced and is cyclically variable. Both induction and cyclic variability can be disadvantages of milk based production compared to methods that rely on processes that occur in a continuous manner throughout the life of an animal, without intervention, and without much variation.
In addition, efforts to produce transgenic products in milk have encountered a variety of other problems. Deleterious effects of endogenous milk constituents, such as proteases, on the desired transgenic product, have been observed. Premature shut-down of lactation has occurred in females expressing mammary specific transgenic proteins. In this regard see, for example, Ebert and Schindler (1993), Transgenic Farm Animals: Progress Report, Theriogenology 39: 121-135. Transgenic mammary gland expression can have deleterious effects on the health of an animal, either directly as a result of the presence in the animal of the transgenic protein or other substance, or as a consequence of induced hyper-lactation necessary to obtain necessary levels of milk production. Finally, while mammary gland-specific expression and secretion into milk appears to modify and process properly a few proteins, it may not do so for others. Inability of mammary gland cells and milk to carry out post-translational processes thus still may prove to be an impediment to commercial production, even if other obstacles can be avoided or overcome.
Perhaps because of such difficulties, mammary gland-specific transgenic expression of particular proteins in milk has not been as widely used for commercial production of transgenic proteins, as might have been expected from the reports on research scale expression. Several other systems have been considered for transgenic production and efforts have been and are being made to use them to produce proteins and, perhaps, other substances. Thus far, each of these systems has problems and/or disadvantages that have prevented their use for commercial production of transgenic proteins or substances, particularly proteins that are produced initially as inactive pro-enzymes that are subject to complex processes of post-translational proteolytic processing and/or modification. There have been several reports on expression of exogenous genes in salivary glands of transgenic animals, for instance. Baum and co-workers reviewed some work in this regard directed to clinical applications, repair of hypofunctional gland parenchyma, in particular, and the production of secretory transgene products for systemic or upper gastrointestinal tract pharmaceutical use (Baum et al. (1999), Critical Reviews in Oral Biology & Medicine 10(3): 276-283. The work described by Baum et al. (1999) related to gene transfer therapy and, apparently, did not aim for commercially advantageous production of proteins, or other substances, in transgenic saliva. Thus, it is relatively uninformative in this regard.
Reports on work directed more specifically to transgenic salivary gland-specific expression have been published by Mikkelsen and co-workers, Larson and co-workers, and Mirels and co-workers (citations follow). Mikkelsen and co-workers reported expression of a Factor VIII-derived polypeptide in saliva of genetically engineered mice. See Mikkelsen et al. (1992), Nature 20(9): 2249-2255. Larson and co-workers also reported salivary gland expression of exogenous gene constructs in transgenic mice. See Larson et al. (1994), Transgenic Research 3(5): 311-316. Mirels and co-workers characterized the genes for rat salivary-gland B1-immunoreactive proteins of adult (and neonatal) rat sublingual and parotid glands (often referred to as the B1-IPs), that also are the major secretory products of rat submandibular gland acinar-cell progenitors. See Mirels et al. (1998), Biochemical Journal 330 (Part 1): 437-444. This work was carried out in species that produce very small quantities of saliva. It is not informative on saliva-specific expression in other mammals, particularly not in ruminants that produce saliva in large volumes. Moreover, apparently only very small amounts of transgenic protein were detected in the saliva, and the work thus is not informative about economically viable and/or commercially advantageous transgenic production of polypeptides and/or proteins and/or other substances in saliva. Apparently, given the lack of further publications, this work has not been pursued, perhaps because of these drawbacks or others.
In sum, present transgenic technologies for pharmaceutical protein production have achieved significant success; but, they have not, as yet, supported commercially advantageous and economically viable production of a marketed pharmaceutical product. Therefore a need exists for improved transgenic animals, methods and technology for commercially advantageous and economically viable production of products for the veterinary and human health care markets, such as pharmaceutical peptides and/or polypeptides and/or proteins, and other substances. Particularly, there is a need for transgenic animals, methods and technology for the commercially advantageous and economically viable production of pharmaceuticals that undergo complex post-translational processing and modification, especially those that cannot be obtained in useful form and quantity by presently available methods of production.